Steroid intermediates

ABSTRACT

Novel compounds prepared by microbial transformation using novel mutants to selectively degrade steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive. These compounds can be used as intermediates to make useful steroids.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 873,101, filed Jan. 30, 1978, now abandoned; which is a division of application Ser. No. 767,369, filed Feb. 10, 1977, now abandoned, which is a division of application Ser. No. 632,250, filed Nov. 17, 1975, which issued as U.S. Pat. No. 4,042,459.

BACKGROUND OF THE INVENTION

The transformation of steroids by microorganisms has been widely studied and documented. Apparently, the earliest such work was by Mamoli and Vercellone in 1937, Ber. 70, 470 and Ber. 70, 2079. They disclosed the reduction of 17-ketosteroids to 17β-hydroxysteroids by fermenting yeast. More recently, Peterson and Murray disclosed the 11α-hydroxylation of progesterone with the fungus Rhizopus nigricans; see. U.S. Pat. No. 2,602,769 (1952). Also recently, Kraychy et al. in U.S. Pat. No. 3,684,657 (1972) discloses the selective microbiological degradation of steroidal 17-alkyls by fermenting a steroid containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3683 to prepare androst-4-ene-3,17-dione, androst-1,4-diene-3,17-dione, and 20α-hydroxymethyl-pregna-1,4-dien-3-one. Even more recently, Marsheck et al. in U.S. Pat. No. 3,759,791 (1973) disclose the selective microbiological preparation of androst-4-ene-3,17-dione by fermenting a steroid of the cholestane or stigmastane series containing at least 8 carbons in the 17-alkyl side chain with Mycobacterium sp. NRRL B-3805.

Compound III of the subject invention has been disclosed by Wang and Sih in 1963 (Biochem. 2: 1238-1243) as an intermediate in the degradation of androst-4-ene-17βol-3-one by Nocardia restrictus. The open ring form of compound I, 7α-methyl-perhydroindanedione-(1,5)-[β-propyl alcohol-(4)], has been disclosed by Schubert et al. in 1964 (Steroids 4: 581-586) as an intermediate in the degradation of progesterone by Mycobacterium smegmatis.

BRIEF SUMMARY OF THE INVENTION

Mutants which are characterized by their ability to selectively degrade steroids with or without 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, and accumulate the following compounds in the fermentation beer:

3aα-H-4α-[3'-propanol]-7aβ-methylhexahydro-1,5-indandione hemiketal ##STR1## hereinafter referred to as Compound I;

3aα-H-4α-[3'-propanal]-5α-hydroxy-7aβ-methylhexahydro-1-indanone hemiacetal ##STR2## hereinafter referred to as Compound II;

3aα-H-4α-[3'-propionic acid]-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone ##STR3## hereinafter referred to as Compound III; and,

3aα-H-4α-[3'-propanol]-5α-hydroxy-7aβ-methylhexahydro-1-indanone ##STR4## hereinafter referred to as Compound IV.

These mutants can be obtained from sterol-degrading microorganisms of the following genera by using the mutation procedures disclosed herein or other mutation procedures: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. A preferred genus is Mycobacterium. Exemplary species of this genus are M. phlei, M. smegmatis, M. rhodochrous, M. mucosum, M. fortuitum, and M. butyricum. Specifically exemplified herein is a novel mutant microorganism, Mycobacterium fortuitum, NRRL B-8128, which is used to selectively degrade steroids with or without 17-alkyl side chain containing from 2 to 10 carbon atoms, inclusive, to the compounds I, II, III and IV. Examples of suitable steroids are sitosterols, cholesterol, stigmasterol, campesterol, and like steroids with 17-alkyl side chains of from 2 to 10 carbon atoms, inclusive, and androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone. These steroids substrates can be in either the pure or crude form.

DETAILED DESCRIPTION OF THE INVENTION

The Microorganisms

Mutants which are characterized by their ability to selectively degrade steroids, as defined above, and accumulate compounds I, II, III and IV in the fermentation beer, can be obtained by mutating sterol-degrading microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. Mycobacterium fortuitum, ATCC 6842, has been mutated, as disclosed herein, to give a novel laboratory mutant microorganism. The 1974 ATCC Catalogue discloses the following alongside the listing of ATCC 6842: "J. C. Cruz 2. Cold abscess. Acta Med. Rio de Janeiro 1:1 (1936). Medium 90 37C". M. fortuitum, ATCC 6842, degrades sterols non-selectively to small molecular weight compounds, e.g. CO₂ +H₂ O. Thus, this microorganism is not suitable as a selective steroid degrader.

Mutation of M. fortuitum, ATCC 6842, using nitrosoguanidine has resulted in the production of a novel mutant which selectively degrades steroids, as defined herein, to produce compounds I, II, III and IV. This mutant microorganism of M. fortuitum has been given the accession number NRRL B-8128, by the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A., where it has been deposited in the permanent collection. A subculture of this microorganism is freely available from this depository by request made thereto. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.

The morphology and drug sensitivities of M. fortuitum, NRRL B-8128, are indistinguishable from that of the parent M. fortuitum, ATCC 6842. Both M. fortuitum cultures are acid-fast non-motile, non-spore-forming bacilli belonging to the family Mycobacteriaceae of the order Actinomycetales. According to Runyon's classification (Runyon, E. H. 1959 Med. Clin. North America 43: 273) it is a nonchromogenic group IV mycobacterium, i.e., it grows rapidly at low temperature to produce nonpigmented colonies on relatively simple media.

M. fortuitum ATCC 6842 and M. fortuitum NRRL B-8128, are clearly distinguishable in their action on steroid molecules. As disclosed above, M. fortuitum ATCC 6842 is a non-selective degrader of steroids, whereas M. fortuitum NRRL B-8128 is a selective degrader. This property of M. fortuitum NRRL B-8128 makes it highly useful, as disclosed herein.

The mutation of M. fortuitum ATCC 6842 to give M. fortuitum NRRL B-8128 was accomplished by the use of nitrosoguanidine. The details of the procedure are described infra. Though mutation procedures are generally known in the art, there is no known art which teaches or even suggests the type of mutants, if any, which might be obtained by use of the subject mutation procedure. Also, though the mutation and transformation procedures, disclosed herein, are detailed for a Mycobacterium, it should be understood that similar or equivalent procedures can be used with microorganisms of the other genera, as disclosed herein.

The Transformation Process

The selective transformation of the subject invention can be effected in a growing culture of M. fortuitum NRRL B-8128 by either adding the selected steroid substrate to the culture during the incubation period, or incorporating it in the nutrient medium prior to inoculation. The steroid can be added singly or in combination with another steroid. The preferred, but not limiting, range of concentration of the steroid in the culture is about 0.1 to about 100 grams per liter. The culture is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, ammonium salts and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

The transformation process can range from about 72 hours to 15 days. The incubation temperature during the transformation process can range from about 25° C. to about 37° C., with 30° C. being preferred. The contents of the transformation vessel are aerated with sterilized air and agitated to facilitate growth of the microorganism, and, thus, enhance the effectiveness of the transformation process.

Upon completion of the transformation process, as evidenced by thin layer chromatography using silica gel plates (E. Merck, Darmstadt) and a solvent system consisting of 2:3 (by volume) ethyl acetate-cyclohexane, the desired transformed steroids are recovered by means well known in the art. For example, the fermentation (transformation) reaction mixture, including the fermentation liquor and cells, can be extracted with a water-immiscible organic solvent for steroids. Suitable solvents are methylene chloride (preferred), chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene and the like.

Alternatively, the fermentation liquor and cells can be first separated by conventional methods, e.g., filtration or centrifugation, and then separately extracted with suitable solvents. The cells can be extracted with either water-miscible or water-immiscible solvents. The fermentation liquor, freed of cells, can be extracted with water-immiscible solvents.

The extracts can be filtered through diatomaceous earth and the filtrate vacuum distilled to dryness. The resulting residue containing the desired transformed steroids then can be dissolved in 10% chloroform in Skellysolve B and chromatographed on silica gel, using Skellysolve B (isomeric hexanes) and mixtures thereof with increasing amounts of ethyl acetate as developing solvent. This procedure elutes compounds I, II and III. Compound IV can be eluted with, for example, 5% methanol in ethyl acetate. Crystalline products can be obtained by use of a solvent, for example, ethyl acetate. The desired transformed steroids can also be obtained from the remaining supernatant upon evaporation of the solvent in the supernatant.

Compounds I, II, III and IV, are useful as intermediates in the chemical synthesis of useful steroids. For example, the subject compounds (90-100 mg) can be oxidized with approximately 40-60 mg of chromic trioxide in approximately 5 ml of acetic acid at room temperature for one hour as disclosed in Bio. 2, at page 1242, to obtain the diketo acid of the formula ##STR5## This diketo acid (0.9 gm) then can be cyclized, according to the procedure disclosed in J.A.C.S. 85: 2135-2137, with 0.1 gm of anhydrous sodium acetate in 40 ml of acetic anhydride at 80°-90° C. for approximately 48 hours to give 0.286 gm of the enol lactone of the formula ##STR6## This enol lactone is Compound I disclosed in Example 1 of U.S. Pat. No. 3,880,884 where R⁷ is oxo. As disclosed in this example, condensation of the enol lactone with a reagent prepared from dimethylmethylphosphonate yields the compound III. This compound is disclosed in Column 8, lines 3-6, as useful to make 19-nor steroids.

The following examples are illustrative of the process and products of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Preparation of Mutant M. fortuitum NRRL B-8128 From M. fortuitum ATCC 6842 (a) Nitrosoguanidine Mutagenesis

Cells of M. fortuitum ATCC 6842 are grown at 28° C. in the following sterile seed medium:

Nutrient Broth (Difco): 8 g/liter

Yeast Extract: 1 g/liter

Sodium Propionate: 0.5 g/liter

Distilled Water, q.s.: 1 liter

The pH is adjusted to 7.0 with 1 N NaOH prior to sterilization at 121° C. for 20 minutes.

The cells are grown to a density of about 5×10⁸ per ml, pelleted by centrifugation, and then washed with an equal volume of sterile 0.1 M sodium citrate, pH 5.6. Washed cells are resuspended in the same volume of citrate buffer, a sample removed for titering (cell count), and nitrosoguanidine added to a final concentration of 50 μg/ml. The cell suspension is incubated at 37° C. in a water bath for 30 minutes, after which a sample is again removed for titering and the remainder centrifuged down and washed with an equal volume of sterile 0.1 M potassium phosphate, pH 7.0. Finally, the cells are resuspended in a sterile minimal salts medium, minus a carbon source, consisting of the following:

NH₄ NO₃ : 1.0 g/liter

K₂ HPO₄ : 0.25 g/liter

MgSO₄.7H₂ O: 0.25 g/liter

NaCl: 0.005 g/liter

FeSO₄.7H₂ O: 0.001 g/liter

Distilled Water, q.s.: 1 liter

The ph is adjusted to 7.0 with 1 N HCl prior to sterilization at 121° C. for 20 minutes. The cells are then plated out to select for mutants.

(b) Selection And Isolation of Mutant M. fortuitum NRRL B-8128

Mutagenized cells, as described above, are diluted and spread onto plates containing a medium consisting of the following (modified from Fraser and Jerrel. 1963. J. Biol. Chem. 205: 291-295):

Glycerol: 10.0 g/liter

Na₂ HPO₄ : 8.4 g/liter

KH₂ PO₄ : 4.5 g/liter

NH₄ Cl: 2.0 g/liter

MgSO₄.7H₂ O: 0.3 g/liter

FeCl₃.6H₂ O: 0.05 g/liter

Distilled Water, q.s.: 1 liter

Agar (15 g/liter) is added, and the medium is autoclaved at 121° C. for 30 minutes and then poured into sterile Petri plates.

Growth on this medium eliminates most nutritional auxotrophs produced by the mutagenesis procedure, e.g. cultures that require vitamins, growth factors, etc. in order to grow on chemically defined medium are eliminated. After incubation at 28° C. for about 7 days, the resulting colonies are replicated to test plates suitable for selecting mutants and then back onto control plates containing the glycerol-based medium. The test plates are prepared as described by Peterson, G. E., H. L. Lewis and J. R. Davis. 1962. "Preparation of uniform dispersions of cholesterol and other water-insoluble carbon sources in agar media." J. Lipid Research 3:275-276. The minimal salts medium in these plates is as described above in section (a) of Example 1. Agar (15 g/liter), and an appropriate carbon source (1.0 g/liter), such as sitosterol or androstenedione (AD), are added and the resulting suspension autoclaved for 30 minutes at 121° C. The sterile, hot mixture is then poured into a sterile blender vessel, blended for several minutes, and then poured into sterile Petri plates. Foaming tends to be a problem in this procedure but can be reduced by blending when the mixture is hot and by flaming the surface of the molten agar plates. In this manner uniform dispersions of water-insoluble carbon sources are obtained which facilitates the preparation of very homogenous but opaque agar plates.

Colonies which grew on the control plates, but not on test plates containing AD as the sole carbon source, are purified by streaking onto nutrient agar plates. After growth at 28° C., individual clones are picked from the nutrient agar plates with sterile toothpicks and retested by inoculating grided plates containing AD as the carbon source. Purified isolates which still exhibit a phenotype different from the parental culture are then evaluated in shake flasks.

(c) Shake Flask Evaluation

Shake flasks (500 ml) contain 100 ml of biotransformation medium consisting of the following ingredients:

Glycerol: 10.0 g/liter

Na₂ HPO₄ : 8.4 g/liter

KH₂ PO₄ : 4.5 g/liter

NH₄ Cl: 2.0 g/liter

MgSO₄.7H₂ O: 0.3 g/liter

FeCl₃.6H₂ O: 0.05 g/liter

Distilled Water, q.s.: 1 liter

Soyflour (1 g/liter) is blended into the medium and then sitosterol (10 g/liter) is also blended into the medium. After the flasks are autoclaved for 20 minutes at 121° C., they are cooled to 28° C. and then inoculated with 10 ml of seed growth prepared as follows:

The purified isolates from part (b) are grown on agar slants at 28° C. A loop of cells taken from a slant is used to inoculate a 500-ml flask containing 100 ml of sterile seed medium consisting of the following ingredients:

Nutrient Broth (Difco): 8 g/liter

Yeast Extract: 1 g/liter

Glycerol: 5 g/liter

Distilled Water, q.s.: 1 liter

The pH is adjusted to 7.0 with 1 N NaOH prior to autoclaving the flasks at 121° C. for 20 minutes. The seed flasks are incubated at 28° C. for 72 hours.

As disclosed above, 10 ml of seed growth is then used to inoculate each 500-ml flask containing 100 ml of sterile transformation medium. The flasks are then incubated at 28° C. to 30° C. on a rotary shaker and sampled at various intervals. Ten ml samples are removed and extracted by shaking with 3 volumes of methylene chloride. Portions of the extracts are analyzed by thin layer chromatography using silica gel and the solvent system described above, i.e., 2:3 (by volume) ethyl acetatecyclohexane, and by gas-liquid chromatography. Evidence of the presence of compounds I, II, III and IV confirms the selective degradation of sitosterol by the novel mutant produced from the parent M. fortuitum ATCC 6842.

EXAMPLE 2 Transformation of Sitosterol to Compounds I, II, III and IV

The medium used is the same as in Example 1c. This medium is sterilized by heating 30 minutes at 121° C., whereupon it is cooled to 30° C. and then inoculated with 10 parts of a seed culture of the mutant mycobacterium M. fortuitum NRRL B-8128, prepared as described in Example 1 (c). The inoculated mixture is incubated at 30° C. for 336 hours with agitation to promote submerged growth. Following incubation, the mixture is extracted with methylene chloride. The extract is filtered through diatomaceous earth and the filtrate is vacuum distilled to dryness. The residue is taken up in 10% chloroform in Skellysolve B and chromatographed on silica gel, using Skellysolve B and mixtures thereof with increasing amounts of ethyl acetate as developing solvent. This procedure elutes compounds I, II and III, Compound IV is eluted from the column with 5% methanol in ethyl acetate. The order of elution of the compounds is I→II→III→IV. The R_(f) values for these compounds on thin layer chromatography (tlc) is as follows (cyclohexane-ethyl acetate; 3:2):

I=0.37

II=0.28

III=0.16

IV=0.05

Appropriate fractions, as determined by tlc, are pooled and evaporated to dryness to afford good yields of compounds I, II, III and IV.

Upon recrystallization from ethyl acetate, compound I, a 1:1 mixture of two epimers, melts at 100°-112° C.;

compound II is an oil;

compound III melts at 122°-124° C.; and

compound IV melts at 148°-150° C.

EXAMPLE 3

By substituting cholesterol for sitosterol in Example 2 there is obtained compounds I, II, III and IV.

EXAMPLE 4

By substituting stigmasterol in Example 2 for sitosterol there is obtained compounds I, II, III and IV.

EXAMPLE 5

By substituting campesterol for sitosterol in Example 2 there is obtained compounds I, II, III and IV.

EXAMPLE 6

By adding a combination of any of the steroids in Examples 2-5, in addition to sitosterol, or in place of sitosterol, in Example 2 there is obtained compounds I, II, III and IV.

EXAMPLE 7

By substituting a sterol-degrading microorganism from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces, in Example 1 for Mycobacterium fortuitum ATCC 6842 there are obtained mutant microorganisms which are characterized by their ability to selectively degrade steroids with or without a 17-alkyl side chain of from 2 to 10 carbon atoms, inclusive, and accumulate compounds I, II, III and IV in the fermentation beer.

EXAMPLE 8

By substituting the mutants obtained in Example 7 for M. fortuitum NRRL B-8128 in Examples 2-6, there is obtained compounds I, II, III and IV.

EXAMPLE 9

By substituting a sterol-degrading microorganism selected from the group consisting of Mycobacterium phlei, M. smegmatis, M, rhodochrous, M. mucosum, and M. butyricum for M. fortuitum ATCC 6842 in Example 1 there are obtained mutant microorganisms which are characterized by their ability to selectively degrade steroids with or without a 17-alkyl side chain of from 2 to 10 carbon atoms, inclusive, and accumulate compounds I, II, III and IV in the fermentation beer.

EXAMPLE 10

By substituting the mutants obtained in Example 9 for M. fortuitum NRRL B-8128 in Examples 2-6, there is obtained compounds I, II, III and IV.

EXAMPLE 11

By substituting a compound selected from the group consisting of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone for sitosterol in Example 2 there is obtained compounds I, II, III and IV.

EXAMPLE 12

By substituting a combination of two or more compounds selected from the group consisting of sitosterol, cholesterol, stigmasterol, androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone, and testosterone for sitosterol in Example 2 there is obtained compounds I, II, III and IV.

EXAMPLE 13

By substituting the mutants obtained in Example 7 for M. fortuitum NRRL B-8128 in Examples 11 and 12, there is obtained compounds I, II, III and IV.

EXAMPLE 14

By substituting the mutants obtained in Example 9 for M. fortuitum NRRL B-8128 in Examples 11 and 12, there is obtained compounds I, II, III and IV. 

We claim:
 1. A compound of the formula ##STR7##
 2. A compound of the formula ##STR8## 